1. Acetylcholinesterase activity in the regenerating forelimb of the adult newt, Triturus, was measured during various stages of development with the Hestrin colorimetric and the Warburg manometric method.

  2. The activity of the enzyme is low during the early formative phases of development. It then increases, the greatest rate of change being in the stage just preceding that of differentiation of muscle. At the time of muscle differentiation the activity levels off and then returns to normal as regeneration draws to a close.

  3. The variation in cholinesterase activity correlates well with the cycle of acetylcholine production during regeneration. The acetylcholine content is very high during early regeneration but then drops to a near normal level as esterase activity rises.

  4. Since the nerve is required for the regeneration process and since the nerve effect is most dramatic when the acetylcholine content is very high, the possibility that acetylcholine may be the agent of the nerve influence is assessed.

  5. The enzymatic activity during regeneration is compared to that observed in embryonic development.

This paper continues the analysis of the role of acetylcholine in the important influence which the nerve exerts on regeneration of a body part in amphibians (reviewed by Singer, 1952, 1959a). Without the nerve, regeneration does not occur. In previous studies it was shown first of all that classical blocking agents of acetylcholine activity—atropine sulphate, procaine hydrochloride, and tetraethylammonium hydroxide—suppress regeneration and delay its resumption for a significant time (Singer, Davis, & Scheuing, 1960).

In a second work which assayed the content of acetylcholine in the developing regenerate, the substance was found in abundance within the growth at all stages of its development (Singer, 1959c). Indeed, during the early formative phases of growth, when development is most dependent upon the nerve, the amount of acetylcholine within the regenerate rose substantially above that of the non-regenerating limb and declined only later during the time of histogenesis. The cycle in content of acetylcholine and the blocking effect of classical acetylcholine inhibitors suggested that acetylcholine may be the agent of the nerve in its control of the early events of regeneration, a conclusion already advanced for planarian regeneration by Welsh (1946). However, there are alternative explanations of our results which were summarized in preliminary fashion in a review by one of us (Singer, 1959c; see also above references). But, whatever the role acetylcholine may play in regeneration of a body part, the fact of the high content raises the additional and important question of the means whereby such a level is attained and maintained. There are a number of possible explanations for the variation in acetylcholine during development, the most salient of which, variation in the content or activity of the well-known hydrolytic enzyme acetylcholinesterase, is herewith analysed by biochemical and histochemical means.

The results show, indeed, that the abundance of acetylcholine within the growth can be correlated inversely with the activity of the esterase. Moreover, the results demonstrate an interesting chemical aspect of regeneration hitherto unsuspected and contribute to our understanding of biochemical mechanisms during regenerative development.

Regenerates of a given stage (stages summarized by Singer, 1952) were removed and homogenized. They were tested for the esterase by one of two methods which employ acetylcholine chloride as the substrate. In one case, the extent of splitting by the esterase was measured gasometrically with a manometer (evolution of carbon dioxide with increase in the acetic acid of the solution), and, in the other, colorimetrically after reacting the residual acetylcholine with hydroxylamine to form hydroxamic acid. The second method was more sensitive and the first served only to confirm the results. Although the first procedure will be described, only brief mention will be made of it in the results.

Bilateral amputations were performed routinely, as in past studies from this laboratory, through the distal third of the upper arm. The operative and postoperative care of animals has also been reported repeatedly. Approximately 10 days after amputation, at a time when regenerative enlargement is first grossly visible at the amputation surface, and thereafter regenerates were selected for assay according to the stage of their development. The growths were removed under a dissecting microscope, care being taken to exclude the proximal adult tissues. Regenerates of the same stage from a number of animals, and often from both sides of the same animal, were homogenized together to provide a measurable amount of enzyme activity. In the younger stages as many as 38 regenerates taken from 19 or more animals were used for determinations.

The method of colorimetric determination

The Hestrin (1949) method yielded a reproducible series of assays of young and old regenerates. The procedure depends on the interaction of acetylcholine with hydroxylamine (H2NOH) as follows:
where R and R′ represent respectively the acetic acid and choline of acetylcholine. Hydroxylamine reacts with acetylcholine stoichiometrically in alkaline pH to yield acethydroxamic acid. Ferric chloride is then added and forms a coloured (purple brown) complex with the acid. After development of the colour the optical density of the solution is read at 540 mμ. The details of the procedure are described adequately in Hestrin’s (1949) original work; but it is necessary to recount some of the procedure here in order to record variations and other items of interest in our determinations of the cholinesterase content of regenerates.

We used 1·75 N sodium hydroxide instead of the recommended 3·5. The pH of the resulting solution was approximately 7·2 instead of 11, and at this pH a minimum of approximately 1 hour was required for the reaction of acetylcholine and hydroxylamine (37·5° C). However, only half the recommended amount of hydrochloric acid sufficed to bring the solution to the pH necessary for the formation of the iron complex. We used 0·5 ml. acetylcholine of a 0·008 M solution and added 0·5 ml. of homogenate solution to attain the final recommended volume for incubation.

The tissue immediately upon weighing was homogenized in a known volume of cold buffer, usually 10 ml. or less. The buffer solution consisted of NaH2PO4.6H2O, 0·05 M; MgCl2.6H2O, 0-02 M; NaCl, 0·1 M; and it was adjusted to a pH of 7 with NaOH solution. This pH is within the optimum for enzymatic hydrolysis of acetylcholine (Hestrin, 1950). The homogenate was centrifuged and the supernatant stored for a brief period at 4° C.

For assay an amount of solution containing 10 mg. of tissue was removed (generally in about 0·1 to 0·2 ml.) and diluted to a volume of 0·5 ml. with 2·8 per cent, fresh gelatin solution, the gelatin presumably increasing the stability of the enzyme (Hestrin, 1950). Then 0·5 ml. of acetylcholine solution was added and the mixture incubated for 2 hours (37·5° C); 2·0 ml. of the alkaline hydroxylamine were then added. The combined solution was incubated at 37·5° C. for 2 additional hours. The time of 2 hours’ incubation and 2 hours’ reaction with hydroxylamine was selected after a number of tests were run at various times. Then concentrated hydrochloric acid (0·5 ml.) was added, followed by ferric chloride, and the volume brought to 5 ml. and read spectrophotometrically. Text-fig. 1 shows the relation between incubation time and enzyme activity expressed as change in light absorption (multiplied by 100) due to declining amount of acetylcholine. The increase in transmission due to hydrolysis of the acetylcholine solution by the homogenate was determined as the difference between the transmission of homogenate and control solutions. Control solutions were treated exactly like the experimental one, adjusted to the same volume, but without added homogenate. The incubation time did not affect the acetylcholine content of the control. In Text-fig. 2 the relation between substrate concentration and enzyme activity is depicted.

Text-fig. 1.

The relation between incubation time of enzyme-substrate and enzyme activity. Activity is expressed as increase in transmittance of an acetylcholine solution resulting from hydrolysis of acetylcholine (see text).

Text-fig. 1.

The relation between incubation time of enzyme-substrate and enzyme activity. Activity is expressed as increase in transmittance of an acetylcholine solution resulting from hydrolysis of acetylcholine (see text).

Text-fig. 2.

The relation between concentration of homogenate and enzyme activity. Activity is expressed as increase in transmittance of an acetylcholine solution resulting from hydrolysis of acetylcholine by a serially diluted homogenate of regenerate tissue (see text).

Text-fig. 2.

The relation between concentration of homogenate and enzyme activity. Activity is expressed as increase in transmittance of an acetylcholine solution resulting from hydrolysis of acetylcholine by a serially diluted homogenate of regenerate tissue (see text).

In addition to the above control, a blank reading was always first made of a solution containing all substances except the enzyme and acetylcholine solutions, which were replaced by water. This reading served as 100 per cent, transmission or 0 per cent, absorption. A turbidity reading was also made of a mixture in which the enzyme solution was inactivated by adding it after hydrochloric acid rather than before. The reduction in transmission due to turbidity was 0·076; it was considered a constant for all runs and it is therefore not included in the calculations.

The homogenates proved enzymatically so active that only a fraction was used for assay; consequently, from 4 to 8 runs were made in each sample. In most instances the readings conformed with one another; but in a few the deviation was great.

The Warburg manometric method

This method is reviewed by Mendel & Hawkins (1950) and only some features are described here. The regenerates were weighed rapidly and then homogenized in 0·9 ml. of cold buffer (NaCl, 0·15 M; MgCl2, 0-04 M; NaHCO3, 0·025 M). The homogenate was transferred to a Warburg vessel (5 ml.); and 0·1 ml. of fresh acetylcholine solution (0 4 M) was introduced into the side arm. The vessel was cooled in a water-bath at 23° C., gassed with nitrogen containing 5 per cent. CO2, and equilibrated for 20 minutes. The solutions were mixed, the vessel shaken (132 times/min.), and readings taken periodically. Controls for non-enzymatic hydrolysis and for homogenate respiration were run simultaneously.

Histochemical studies of cholinesterase

Attempts were made to localize acetylcholinesterase within tissues of the regenerate at various stages of development. Studies were made on 22 regenerates distributed as follows among the stages: 8 early regenerates, 3 medium, 4 late to palette, and 7 advanced palette or early digital stages (see stages of Singer, 1952). The cholinesterase was visualized according to the method of Koelle (1950) as modified by Chessick (1954). The limb-stump and regenerate were removed without anaesthesia. The bone was then teased from the stump and the tissue fixed overnight (for about 8 hours) in cold buffered formalin (1 part 40 per cent, formaldehyde to 9 parts M/15 phosphate buffer of pH 6·5·6·8). The specimen was washed for 10-30 minutes and impregnated in 10 per cent, gelatin (4 hours at 37° C.). It was fixed again in formalin for 10·30 minutes. Frozen sections were made at 10·25 μ. Staining, mounting, and dehydration procedures are described by Koelle (1950). Sections were generally counterstained with 0 · 5 per cent, methylene blue. The site of activity was visualized by the precipitation of copper thiocholine; subsequent treatment with ammonium sulphide converted the crystals to copper sulphide (Malmgren & Sylvén, 1955). A positive reaction was a golden tan to dark-brown colour. In the histochemical tests the incubation media and procedures suggested by Koelle (1950) were used to identify all cholinesterases or, selectively, specific acetylcholinesterase and non-specific cholinesterase. According to these procedures, non-specific cholinesterase is inhibited by low concentrations (10−6 M) of di-isopropylfluoro-phosphate (DFP), whereas specific acetylcholinesterase requires much higher concentrations.

The tests were as follows:

Control slides were incubated in DFP (10−3 M) for 30 minutes and then tested for ‘all cholinesterases’ (A above). The DFP in this concentration destroys all cholinesterases, including specific acetylcholinesterase.

The acetylcholinesterase of normal and regenerating forelimbs

Table 1 lists the results of enzymological tests of the activity of cholinesterase in normal and regenerating limbs of various ages. In Text-fig. 3 the average activity is plotted against the age in days and approximate stage of development. The average activity is expressed per unit of dry weight of regenerate. The reason for expressing the results in this way has already been given in a preceding work on the acetylcholine content of the regenerate (Singer, 1959c), but should be touched upon here. In the present assay for cholinesterase activity the determinations were always made on 10 mg. of tissue. However, the young regenerate is quite edematous and the water content is substantially higher than the older one. Consequently, the enzyme activity was diluted by the abundant fluid. If the activity is expressed in dry weight the influence of water content on the activity can be controlled and the various stages can now be compared directly with one another.

Table 1.

The activity of cholinesterase

The activity of cholinesterase
The activity of cholinesterase
Text-fig. 3.

The activity of the enzyme is expressed as loss in light absorption of the ferric hydroxamic acid complex due to hydrolysis of the acetylcholine substrate. All values are based on dry weight × 10 (see Table 1, last column). Sketches are included of representative regenerative stages, from left to right: early bud, medium bud, late bud, palette, early digital, and medium digital. The age of each stage is an average one based on many previous experiences. Non-stippled area is the amputation stump; the border is the original amputation line. Sketches about × 3 actual size.

Text-fig. 3.

The activity of the enzyme is expressed as loss in light absorption of the ferric hydroxamic acid complex due to hydrolysis of the acetylcholine substrate. All values are based on dry weight × 10 (see Table 1, last column). Sketches are included of representative regenerative stages, from left to right: early bud, medium bud, late bud, palette, early digital, and medium digital. The age of each stage is an average one based on many previous experiences. Non-stippled area is the amputation stump; the border is the original amputation line. Sketches about × 3 actual size.

The dry weight of the regenerate at each stage of development was reported in a previous work (Singer, 1959c). Since there was a direct relation between optical transmission and cholinesterase activity (Text-fig. 2), the results could be recalculated to read transmission per 10 mg. dry weight instead of 10 mg. wet weight (see Table 1). The final value was multiplied by 10 in order to obtain a convenient value for plotting (Text-fig. 3). Therefore, the plot on Text-fig. 3 represents the activity of the enzyme per 100 mg. of regenerate. The corrections for water content were based on the following percentages of dry weight per individual stage taken from Singer (1959c): normal forelimb without bone, 23·4 per cent.; early regenerate bud, 12·8 per cent.; medium bud, 12·8 per cent.; late bud, 14·8 per cent.; palette, 141 per cent.; and early digital regenerate, 17·8 per cent. An example of the calculations is as follows for the early regenerate bud: 0·043 (the measured increase in transmission due to enzyme hydrolysis by the 10-mg. sample of wet tissue) multiplied by the fraction 100/12·8 yields the expected increase in light transmission due to enzyme activity in a sample representing 100 mg. dried tissue.

The turbidity of the homogenate decreased the extent of light transmission. Consequently, each determination should be higher, roughly according to the content of dried tissue; in the case of the early regenerate bud the additional amount should be about half of that for the normal limb, the turbidity reading of which was 0·076.

The results depicted in Table 1 and Text-fig. 3 show that the activity of acetylcholinesterase is very low during the early formative phases of regeneration, but then climbs rapidly during the stages of the late bud and during early differentiation. In the early regenerate bud and perhaps in a still earlier stage, there is a small but measurable amount of cholinesterase. Wound tissues in the early post-amputation days were not measured. Beyond the stage of the early bud the enzyme activity per unit of tissue increases slowly at first and then more rapidly as differentiation of muscle and other tissue sets in. During the latter time the enzyme activity tends to rise above that of normal; still later, it presumably falls and eventually levels off near the normal value.

We were unable to measure the enzyme activity in the early phases of regeneration with the Warburg method. However, in the later stages to which the technique was adequately sensitive, there was a rise in activity which paralleled that already described with the Hestrin method.

Histochemical observations on cholinesterase of regenerating limbs

A considerable amount of adult tissue of the stump was removed with the regenerate and served as the normal control. The stump tissues always showed a marked positive reaction for all cholinesterases (Plate, fig. A) and for specific cholinesterase (Plate, fig. B). Most of the positive reaction was localized in muscle which stained a deep golden brown and showed distinct striations. The nerve was also stained but much more faintly. In the control sections previously treated with a high concentration of DFP the reaction was entirely absent (Plate, fig.D).

We detected no reaction for any of the cholinesterases in the early, medium, and late regenerate buds (Plate, figs. A, B). The regenerate area was negative whereas the stump was strikingly stained. In the late palette stage a faint reaction occurred near the base of the regenerate where muscle first differentiates. It may be noted here that there are two types of muscle formed in the regenerate of the adult limb. The first is of muscle transected during amputation. This muscle undergoes sarcolysis at its end and releases many cells which extend in a stream into the wound area. During the palette stage cells of this stream begin to differentiate into muscle fibres; and these fibres give the first cholinesterase reaction. The second type of muscle is formed within the regenerate without morphological connexion to previous muscle and represents muscle removed completely during amputation. It appears later than the first type and is visible in the very early digital phase as sarcoblasts associated with delicate cross-striated fibrillae. By the time of the differentiation of this muscle, the specific cholinesterase reaction of the regenerate was almost as intense as that for the stump (Plate, fig. C); an adjacent control section treated with a high concentration of DFP is shown in the Plate, fig. D. Nerve trunks of the regenerate, in contrast to muscle, stained very lightly. In summary, the results of histochemical study support those of the enzymological assays, namely that cholinesterase activity is quite low or relatively absent in the early stages of growth; it then increases sharply during the time of differentiation. The histochemical observations also show that the major part of the activity is located in the muscle.

The results show that the activity of acetylcholinesterase is low during the phases of formation, accumulation, and rapid growth of the regenerate. It rises sharply during morphogenesis and histogenesis when it finally approaches (and may even exceed) the activity of esterase in the adult forelimb. The enzymological assay is supported by the histochemical one.

In a preceding paper (Singer, 1959c) the acetylcholine content of the regenerate was shown to be relatively high in the early stages of regeneration. Moreover, during the phases when mesenchymatous cells first appear among the distal wound tissues and accumulate to form the blastema, as well as when the blastema grows rapidly, the acetylcholine content rises high above that in nonregenerating tissues. Later, at the time of morphogenesis and histogenesis, acetylcholine declines and approaches that of the normal.

A good reason for the rise and decline of acetylcholine emerges from the present results. In Text-fig. 4 the cholinesterase activity and the acetylcholine content of the regenerate, scaled for direct comparison, are plotted for different stages of development. The graph shows an apparently causal relation between the acetylcholine-splitting enzyme and the acetylcholine content. When the activity of the enzyme is low, the rate of hydrolysis of acetylcholine is also low and the substance accumulates in higher quantities than in adult tissues. As the enzyme activity increases substantially the amount of acetylcholine per unit of regenerate declines until it finally approaches the normal value at the time of maximum cholinesterase content. There may also be other reasons for the accumulation of acetylcholine within the early growth, such as great abundance of nerve fibres in the regenerating tissue and greater choline acetylase activity.

Text-fig. 4.

Comparison of acetylcholine content and acetylcholinesterase activity in regenerating forelimbs of various ages and stages (see legend for Fig. 3 of this paper; and for Fig. 2, Singer, 1959c; also Singer, 1959c).

Text-fig. 4.

Comparison of acetylcholine content and acetylcholinesterase activity in regenerating forelimbs of various ages and stages (see legend for Fig. 3 of this paper; and for Fig. 2, Singer, 1959c; also Singer, 1959c).

The high level of acetylcholine and the low activity of cholinesterase occur during the phases of regeneration when the influence of the nerve upon the growth is most dramatic. It is worth recounting at this time the salient facts of this influence. If the adult stump is denervated at the time of amputation, mesenchymatous cells of regeneration do not appear (reviewed by Singer, 1952). If the regenerate is denervated later, during the phase of blastema formation, then, except for histogenesis of the individual tissues in the wound region, there is no further enlargement and regeneration of the missing parts (Singer & Craven, 1948). Once the regenerate bud has grown and contains many cells it will rearrange its substance and elongate to form a small limb in the absence of the nerve. Consequently, the phases of cellular appearance and accumulation and of subsequent rapid growth require the presence of the nerve; and it is during these phases that the cholinesterase activity is lowest and the content of acetylcholine is highest.

The question must now be asked: is acetylcholine the agent of the nerve in its important action in regeneration of a body part? The causal relation between the acetylcholine amount within the growth and the activity of cholinesterase, which ensures a high level of the substance during the time when regeneration is most dependent upon the nerve, suggests an affirmative answer to this question. Such a conclusion seems to be supported further by the fact that atropine and other drugs which interfere with acetylcholine activity also suppress regeneration (Singer, Davis, & Scheuing, 1960). An affirmative conclusion would mean that the acetylcholine mechanism of the nerve, which is important in transmission or conduction of the nerve impulse, also serves as the ‘trophic’ mechanism. However, the variation in acetylcholine may be merely coincidental to the phases of regeneration most dependent upon the nerve; or it may be of other significance for regeneration. Indeed, in the light of information now emerging in our laboratory it is questionable that acetylcholine is the nervous agent of growth. This information is summarized in a review presented before the Growth Society (Singer, 1959b).

There are other possible actions of acetylcholine than that of a ‘trophic agent’. It is said to be a surface agent which alters the permeability of protoplasmic membranes (Welsh & Taub, 1948). In this way it may function in regeneration by providing for the passage of important substances into cells. Another thought is that acetylcholine may serve as an important metabolic agent (Gerard, 1950) and indeed as a coenzyme (Welsh, 1948). In experiments from our laboratory (for preliminary summary see Singer, 1959b), we have observed regeneration under circumstances to which the variations in acetylcholine content could not be correlated. It is, therefore, conceivable that acetylcholine has no developmental significance; it may become of functional value only when anatomical structure is differentiated.

Aside from its possible interest in regeneration studies, it is of some importance and interest to compare the cholinesterase content of the developing regenerate with that of the embryo. A number of enzymological and histochemical studies have been made on cholinesterase activity in the amphibian embryo (Sawyer, 1943 a, b, 1955; Boell, 1948; Boell & Shen, 1944, 1949, 1950; Boell, Greenfield, & Shen, 1955). Cholinesterase appears in significant quantity at the time of first motility when the neuromuscular apparatus is differentiating. It is found at first in the spinal cord in a region where the early reflexes are initiated, and it appears thence (in order) in the hindbrain, midbrain, and forebrain. Therefore, during the early formative stages of embryonic development, there is little activity of the esterase. Later, associated with functional differentiation, there is a rise in cholinesterase which is much greater than the increase in some other enzyme systems or in the protein content of the nervous tissues (Boell, Greenberg, & Shen, 1955).

In the development of cholinesterase activity the regenerate resembles the embryo. The cholinesterase activity is low in the early formative stages of regrowth during the periods of so-called dedifferentiation and of cellular accumulation. It climbs slowly during the subsequent phase of rapid cellular multiplication and regenerate enlargement. At about the time of onset of morphogenesis, the activity rises sharply, and then approaches the value of adult tissues during the stage of digit formation. The sharpest rate of increase coincides with the time of early myogenesis. Thus, in the regenerate as in the embryo, the greatest increase in the activity of cholinesterase is correlated directly with the differentiation of muscle and therefore with the first signs of motility.

It seems likely that what little cholinesterase activity exists in the early stages is confined to the nerve fibres, and that the mesenchymatous cells of regeneration which constitute the blastema are relatively devoid of the active enzyme. Some of these cells are said to arise by a process of morphological dedifferentiation from muscle; assuming this to be the case, then dedifferentiation affects the activity of this enzyme in addition to degrading morphological structure. Later, at the time of histogenesis, differentiation or the reactivation of the enzyme occurs. Boell, Greenfield, & Shen (1955) showed that the development of enzyme activity during embryogenesis was about 10 times that of protein and consequently much more rapid than the development of structure. Protein determinations have not been made for the regenerate, but a rough comparison of the increase in dry weight and in cholinesterase activity suggests that a similar relation obtains for the regenerate. The increase in cholinesterase activity proceeds at a greater rate than increase in substance of the regenerate, especially during the period preceding and accompanying functional maturation and differentiation of muscle elements.

Although the development of cholinesterase activity in the regenerate resembles that in the embryo, it also differs from it. In the early premotile stages of Amblystoma (stage 36 of Harrison) cholinesterase activity is too low to be detected. It then appears suddenly and increases rapidly, coinciding with the first signs of motility. In the case of the regenerate, on the other hand, there is a detectable amount of activity in the earliest stage measured, namely, at a time when the blastema is just being formed. Consequently, regenerative—unlike embryonic—development occurs presumably from the start in the presence of some cholinesterase. The location of the enzyme is not known for certain at the moment but preliminary experiments on denervated regenerates seem to point to the nerve fibres themselves as the source of the activity. Nerve fibres appear among wound tissues within 2 or 3 days after amputation. They multiply rapidly and invade all tissues of the regenerate during subsequent days of development (Singer, 1949). Therefore, in the regenerate, detectable cholinesterase activity precedes by many days and stages of growth the first signs of motility and muscle differentiation, although the greatest rate of increase does coincide with functional and structural differentiation. The small amount of cholinesterase of the early regenerate is apparently not a prerequisite for later differentiation. Even after denervation of the regenerate and, therefore, after the loss of nerve cholinesterase, differentiation can occur only provided that the regenerate is of an adequate size at the time of nerve loss (Singer, 1952).

L’activité acétylcholinestérasique dans la régénération du membre antérieur du Triton adulte, Triturus

  1. L’activité acétylcholinestérasique au cours de la régénération du membre antérieur du Triton adulte, Triturus, a été mesurée à différents stades du développement à l’aide de la méthode colorimétrique d’Hestrin et de la méthode mano-métrique de Warburg.

  2. L’activité de l’enzyme est faible pendant les premières phases du développement. Elle augmente ensuite, le taux le plus élevé étant au stade qui précède immédiatement la différenciation du muscle. Au moment de la différenciation du muscle, elle cesse d’augmenter et revient à l’activité normale quand la régénération touche à sa fin.

  3. La variation de l’activité cholinestérasique est en corrélation avec le cycle de la production d’acétylcholine pendant la régénération. Le contenu en acétylcholine est très élevé pendant le début de la régénération mais ensuite il tombe à un niveau presque normal quand l’activité estérasique s’élève.

  4. Comme le système nerveux est nécessaire au processus de régénération et que l’effet du nerf est le plus marqué quand la teneur en acétylcholine est la plus élevée, l’hypothèse suivant laquelle l’acétylcholine est l’agent de l’influence nerveuse est discutée.

  5. L’activité enzymatique durant la régénération est comparée à celle que l’on observe dans le développement embryonnaire.

The study was supported by grants from the American Cancer Society and the National Institutes of Health, Public Health Service.

Boell
,
E. J.
(
1948
).
Biochemical differentiation during amphibian development
.
Ann. N.Y. Acad. Sci
.
49
,
773
800
.
Boell
,
E. J.
,
Greenfield
,
P.
, &
Shen
,
S. C.
(
1955
).
Development of cholinesterase in the optic lobes of the frog (Rana pipiens)
.
J. exp. Zool
.
129
,
415
51
.
Boell
,
E. J.
, &
Shen
,
S. C.
(
1944
).
Functional differentiation in embryonic development. I. Cholinesterase activity of induced neural structure in Amblystoma punctatum
.
J. exp. Zool
.
97
,
21
41
.
Boell
,
E. J.
, &
Shen
,
S. C.
(
1949
).
Experimental modification of cholinesterase development in the midbrain of Amblystoma punctatum
.
Anat. Rec
.
105
,
490
.
Boell
,
E. J.
, &
Shen
,
S. C.
(
1950
).
Development of cholinesterase in the central nervous system of Amblystoma punctatum
.
J. exp. Zool
.
113
,
583
600
.
Chessick
,
R. D.
(
1954
).
The histochemical specificity of cholinesterase
.
J. Histochem. Cytochem
.
2
,
258
73
.
Gerard
,
R. W.
(
1950
).
The acetylcholine system in neural function
.
Recent Progress in Hormone Research
,
5
,
37
61
.
Hestrin
,
S.
(
1949
).
The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application
.
J. biol. Chem
.
180
,
249
61
.
Hestrin
,
S.
(
1950
).
Acylation reactions mediated by purified acetylcholine esterase. II
.
Biochim. biophys. Acta
,
4
,
310
21
.
Koelle
,
G. B.
(
1950
).
The histochemical differentiation of types of cholinesterases and their localizations in tissues of the cat
.
J. Pharmacol
.
100
,
158
79
.
Malmgren
,
H.
, &
Sylvén
,
B.
(
1955
).
On the chemistry of the thiocholine method of Koelle
.
J. Histochem. Cytochem
.
3
,
441
8
.
Mendel
,
B.
, &
Hawkins
,
R. D.
(
1950
).
Estimation of the cholinesterases
.
Meth. med. Res
.
3
,
107
15
.
Sawyer
,
C. H.
(
1943a
).
Cholinesterase and the behaviour problem in Amblystoma. I. The relationship between the development of the enzymes and early motility. II. The effects of inhibiting cholinesterase
.
J. exp. Zool
.
92
,
1
29
.
Sawyer
,
C. H.
(
1943b
).
III. The distribution of cholinesterase in nerve and muscle throughout development. IV. Cholinesterase in nerveless muscle
.
J. exp. Zool
.
94
,
1
31
.
Sawyer
,
C. H.
(
1955
).
Further experiments on cholinesterase and reflex activity in Amblystoma larvae
.
J. exp. Zool
.
129
,
561
78
.
Singer
,
M.
(
1949
).
The invasion of the epidermis of the regenerating forelimb of the urodele, Trituras by nerve fibers
.
J. exp. Zool
.
111
,
189
210
.
Singer
,
M.
(
1952
).
The influence of the nerve in regeneration of the amphibian extremity
.
Quart. Rev. Biol
.
27
,
169
200
.
Singer
,
M.
(
1959a
).
The influence of nerves on regeneration
.
In Regeneration in Vertebrates
, ed.
C. S.
Thornton
, pp.
59
80
.
University of Chicago Press
.
Singer
,
M.
(
1959b
).
Nervous mechanisms in the regeneration of body parts in vertebrates
.
In 18th symposium of the Society for Study of Development and Growth
, ed.
D.
Rudnick
.
Singer
,
M.
(
1959c
).
The acetylcholine content of the normal forelimb regenerate of the adult newt, Trit urns
.
Devel. Biol
.
1
,
603
20
.
Singer
,
M.
&
Craven
,
L.
(
1948
).
The growth and morphogenesis of the regenerating forelimb of adult Trituras following denervation at various stages of development
.
J. exp. Zool
.
108
,
279
308
.
Davis
,
M. H.
, &
Scheuing
,
M. R.
(
1960
).
The influence of atropine and other neuropharma-cological substances on regeneration of the forelimb in the adult urodele, Trituras
.
J. exp. Zool
. (In press.)
Welsh
,
J. H.
(
1946
).
Evidence of a trophic action of acetylcholine in a planarian
.
Anat. Rec
.
94
,
421
.
Welsh
,
J. H.
(
1948
).
IV. Concerning the mode of action of acetylcholine
.
Bull. Johns Hopkins Univ
.
83
,
568
79
.
Welsh
,
J. H.
&
Taub
,
R.
(
1948
).
The action of choline and related compounds on the heart of Venus mercenaria
.
Biol. Bull
.
95
,
346
53
.

Figs. A-D. Sections of regenerates of various ages tested histochemically for cholinesterase activity.

Fig. A. Early bud and amputation stump showing distribution of ‘all’ cholinesterases. The reaction is negative (light staining) in the regenerating tissues (−) but highly positive (dark staining) in muscles of stump ( + ). Nerves show faint positive reactions. The central cavity is the space normally occupied by the humerus, × 35.

Fig. B. Very late bud or early palette and amputation stump showing distribution of specific acetylcholinesterase. There is relatively little reaction (−) in the entire regenerate area; but a strong positive reaction (+) in the stump, × 27.

Fig. C. Digital regenerate showing specific acetylcholinesterase. Note the strong reaction ( + ) among the muscles of the regenerate, × 21.

Fig. D. Section adjacent to that of fig. C. Section treated with di-isopropylfluorophosphate (DFP) (10−3 M), then tested for acetylcholinesterase. Note complete absence of reaction in regenerate and stump, × 21.

Figs. A-D. Sections of regenerates of various ages tested histochemically for cholinesterase activity.

Fig. A. Early bud and amputation stump showing distribution of ‘all’ cholinesterases. The reaction is negative (light staining) in the regenerating tissues (−) but highly positive (dark staining) in muscles of stump ( + ). Nerves show faint positive reactions. The central cavity is the space normally occupied by the humerus, × 35.

Fig. B. Very late bud or early palette and amputation stump showing distribution of specific acetylcholinesterase. There is relatively little reaction (−) in the entire regenerate area; but a strong positive reaction (+) in the stump, × 27.

Fig. C. Digital regenerate showing specific acetylcholinesterase. Note the strong reaction ( + ) among the muscles of the regenerate, × 21.

Fig. D. Section adjacent to that of fig. C. Section treated with di-isopropylfluorophosphate (DFP) (10−3 M), then tested for acetylcholinesterase. Note complete absence of reaction in regenerate and stump, × 21.